Enzyme stabilization in electrochemical sensors

ABSTRACT

The present invention relates to a composition for forming an electrode, an electrochemical sensor comprising the same, and a method for determining an analyte using the electrochemical sensor.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/308,049, filed Jun. 18, 2014, which is a continuation of U.S. patentapplication Ser. No. 13/296,871, filed Nov. 15, 2011, which is acontinuation of International Application No. PCT/EP2010/056668, filedMay 14, 2010, which claims the benefit and priority of European PatentApplication No. 09160421.5, filed May 15, 2009. The entire disclosuresof the above applications are incorporated herein by reference.

BACKGROUND

The present invention relates to a composition for forming an electrode,an electrochemical sensor comprising the same, and a method fordetermining an analyte using the electrochemical sensor.

Measuring systems for biochemical analysis are important components ofclinically relevant analytical methods. This primarily concerns themeasurement of analytes which can be directly or indirectly determinedwith the aid of enzymes. Determination of the concentration ofclinically useful parameters is generally carried out using in vitroanalytical systems. However, when determining analytes which show asignificant change in their concentration during the course of the dayan in vitro analysis is inappropriate due to limited temporal resolutionand the difficulties encountered with sampling.

In this case, biosensors, i.e., measuring systems equipped withbiological components, which allow a repeated measurement of the analyteeither continuously or discontinuously and which can be used ex vivo aswell as in vivo have proven to be particularly suitable for themeasurement of analytes. Ex vivo biosensors are typically used inflow-through cells whereas in vivo biosensors can, for example, beimplanted into subcutaneous fatty tissue. In this connection onedistinguishes between transcutaneous implants which are only introducedinto the tissue for a short period and are in direct contact with ameasuring device located on the skin, and full implants which areinserted surgically into the tissue together with a measuring device.

Electrochemical biosensors which comprise an enzyme as a biologicalcomponent contain the enzyme in or on the working electrode in whichcase for example the analyte can serve as a substrate for the enzyme andcan be physicochemically altered (e.g. oxidized) with the aid of thisenzyme. The electrical measuring signal generated by the flow ofelectrons released during conversion of the analyte on the workingelectrode correlates with the concentration of the measured analyte suchthat the electrical measuring signal can be used to determine thepresence and/or the amount of the analyte in the sample.

In practice, a working electrode must fulfill a number of requirementsin order to be suitable in electrochemical sensors:

The working electrode should have a low contact resistance and henceshould be highly conductive.

The working electrode should not comprise any components which areelectrochemically converted in the selected polarisation voltage. Thiscan be accomplished by a suitable choice of binders and fillers.

The electrochemically active surface area of the working electrode hasto be kept constant over its entire period of operation. For thispurpose, a reduction of the surface area due to the adsorption ofcomponents of the surrounding fluid has to be avoided. This is generallyaffected by applying one or several polymer coatings, which are highlybiocompatible.

The electrochemical reaction of the conversion product of the enzymaticreaction should be effected at a low overpotential in order to minimizethe decomposition voltage and hence enable a specific conversion of theparameter. For this purpose, a fast transfer of electrons from theprosthetic group of the enzyme to the diverting electrode is to beprovided.

The working electrode should comprise a sufficient amount ofanalyte-specific enzyme having sufficient and constant activity in orderto guarantee that the enzymatic reaction superposed to theelectrochemical conversion is not limited by the available enzymaticactivity, but by the available amount of analyte. In other words, thesensitivity has to be maintained throughout the entire period ofoperation. A diffusion of the enzyme from the working electrode into thesurrounding tissue has to be avoided, also for the reason of a possibletoxicity of the enzyme. Finally, it has to be provided for that theenzymatic activity does not fall below a predetermined limit duringstorage.

A plurality of electrode compositions is known for minimizingoverpotentials. As regards H₂O₂, the oxidation potential can be reduced,for example, by 450 mV by using rhodium- and glucose oxidase-coatedcarbon fibres as compared to carbon fibres coated with glucose oxidasealone (Wang et al., Analytical Chemistry (1992), 64, 456-459). A methodwhich is simpler to realize is described in EP 0 603 154 A2, whichdocument provides an electrode composite produced by thoroughly mixingoxides and/or hydroxides of elements of the 4th period of the periodictable with graphite and a binder, leading to a reduction in overvoltageof the anodic H₂O₂ oxidation by >200 mV.

In addition to electrically nonconductive and/or semiconductive metaloxides generally introduced into electrode composites, electricallyconductive electrocatalysts such as carbon nanotubes are known, which,due to their small size, may be arranged in proximity to the prostheticcentre of the enzyme, have a high electric conductivity and enable anefficient transfer of electrons (Wang et al., Analyst (2003), 128,1382-1385; Wang et al., Analyst (2004), 129, 1-2; Wang et al., AnalyticaChimica Acta (2005), 539, 209-213; Shobha Jeykumari et al., Biosensorsand Bioelectronics (2008), 23, 1404-1411). Due to their high surfacearea, small amounts of nanotubes are sufficient to obtain a reduction ofthe decomposition potential (US 2006/0021881 A1).

Different measures are known in the art in order to provide for aconstant enzyme activity. One possibility to avoid enzyme diffusion fromthe surface of the working electrode into the environment is to providethe working electrode with a suitable coating, e.g. a cover membrane.However, the use of such coatings in electrochemical sensors isassociated with certain problems such as the necessity to depositpinhole-free membranes. Secondly, the cover membrane must be depositedwith a highly reproducible layer thickness for mass transfer limitedsystems. This requirement represents an extensive restriction ofpossible coatings since the realization of very thin layers exhibiting asufficiently high barrier to mass transfer is difficult to realize.

Moreover, electrochemical sensors which are used to determine differentanalytes must usually also contain different cover membranes in order toprovide different mass transfer rates of the substrate and co-substrateto the electrode. At the same time it must be ensured that the covermembranes are highly biocompatible for in vivo applications. Since eventhe smallest defects in the membrane are sufficient to result in ableeding of the enzyme from the electrode into the environment, anenormous amount of quality control is necessary especially in the caseof in vivo biosensors, resulting in considerable technical requirementsand increased production costs.

Alternatively, the extent of enzyme bleeding can be reduced byimmobilizing the enzyme in the electrode matrix of the working electrodewhich has led to an intensive search for suitable immobilization methodsfor enzymes in electrochemical biosensors. In practice, the enzyme mayeither be coupled to one or more paste components in a chemicallycovalent manner or be inserted physically into a composite so that theenzyme is adsorptively bonded to one or several paste components and/oris incorporated therein.

As regards adsorptive immobilization, Rege et al. (Nano Letters (2003),3, 829-832) disclose an electrode composite comprising single-wallcarbon nanotubes (SWCNT) and/or graphite as conductive fillers and PMMAas binder, wherein chymotrypsin is physically retained. In this context,it was found out that SWCNT-containing composites show less enzymebleeding than graphite-containing ones, which seems to be due to ahigher surface energy of the SWCNTs as compared to graphite.

Tang et al. (Analytical Biochemistry (2004), 331, 89-97) showed that byusing a CNT-electrode, onto which Pt particles were electrochemicallydeposited and which was adsorptively modified with glucose oxidase, thelong-term stability of glucose oxidase can be significantly increased ascompared to a conventional graphite electrode.

Tsai et al. (Langmuir (2005), 21, 3653-3658) disclose a stable glucosesensor which comprises a carbon electrode coated with a compositecontaining multi-wall carbon nanotubes (MWCNTs), Nafion and glucoseoxidase. The immobilizing effect of the matrix is referred to theelectrostatic interaction of negatively charged MWCNTs and Nafion on theone hand, and positively charged glucose oxidase on the other hand.

Guan et al. (Biosensors and Bioelectronics (2005), 21, 508-512) realizea glucose sensor by dispensing a MWCNT-containing suspension and a GOD-and a ferricyanide-containing solution onto a screenprinted carbonelectrode. An increased linearity of the response curve was observed andattributed to an increased electron transfer rate of the MWCNTs ascompared to that of a conventional carbon electrode.

Kurusu et al. (Analytical Letters (2006), 39, 903-911), finally,disclose that the use of an electrode comprising a mixture of MWCNT, GODand mineral oil leads to a significant reduction of the oxidativedecomposition voltage of H₂O₂.

However, adsorptive immobilization procedures suffer from a number ofproblems. A major disadvantage of physical immobilization is thedependency of the binding constant on the composition of the mediumsurrounding the electrode, requiring a barrier membrane to preventenzyme leakage.

In particular, however, physical coupling makes heavy demands on thereproducibility of the effective surface of the working electrode and,thus, on the production thereof. As described above, adsorptiveimmobilization either requires an application of enzyme-containingsolutions and/or suspensions onto the surface of a prefabricated workingelectrode or an introduction of enzyme-containing solutions and/orsuspensions into an electrode composite. The dispensing application ofenzyme-containing solutions and/or suspensions onto the surface of aworking electrode, however, has the disadvantage that an addition ofsmall volumes of enzyme solution, for example a volume in the range ofnanoliters, makes high demands on the precision of an automated dosingapparatus. Furthermore, the distribution of enzyme on the surface of theworking electrode and transfer of enzyme into the pores of the workingelectrode depends on the topography and the energy of the electrode'ssurface, which is difficult to reproduce.

In view of the above, the admixture of enzymes into an electrodecomposite is to be preferred. However, a loss of the effective enzymeactivity caused by shearing, the influence of solvents and thermalimpact cannot be avoided due to the requirement of a homogeneousdistribution of enzymes in mostly hydrophobic composites. Moreover,restrictions with respect to overall paste conductivity and adhesiononto the substrate have to be taken into account since specificrheological characteristics are required for the deposition of theelectrode paste, while a constant consistency of the paste afterhomogeneous distribution of a dry enzyme in the composite has to beprovided.

As an alternative, the enzyme may be introduced into the composite in anaqueous solution or in a suspension in order to minimize denaturation.This, however, brings about the necessity of a subsequent removal ofsolvent or suspension agent so that the composite cannot be supposed tohave constant rheological characteristics over the production period.

Hence, in view of the disadvantages of an adsorptive immobilizationthere is thus a concrete need to immobilize enzymes in electrochemicalbiosensors by covalent bonds to at least one component of the electrodematrix.

EP 0 247 850 A1 discloses biosensors for the amperometric detection ofan analyte. These sensors contain electrodes with immobilized enzymeswhich are immobilized or adsorbed onto the surface of an electricallyconducting support where the support consists of a platinized porouslayer of resin-bound carbon or graphite particles or contains such alayer. For this purpose, electrodes made of platinized graphite and apolymeric binding agent are firstly prepared and these are subsequentlybrought into contact with the enzyme. In this case, the enzyme isimmobilized either by adsorption to the electrode surface or by couplingit to the polymeric binding agent using suitable reagents.

Amperometric biosensors with electrodes comprising an enzyme immobilizedor adsorbed onto or into an electrically conducting, porous electrodematerial are also described in EP 0 603 154 A2. In order to produce theenzyme electrodes, an oxide or oxide hydrate of a transition metal ofthe fourth period, such as for example manganese dioxide, acting as acatalyst is worked into a paste together with graphite and anon-conducting polymeric binding agent, and the porous electrodematerial obtained after drying the paste is brought into contact withthe enzyme in a second step. The enzyme can be immobilized on or in theporous electrode material by using a cross-linking reagent such asglutaraldehyde.

A major disadvantage of the electrochemical biosensors described in EP 0247 850 A1 and EP 0 603 154 A2 is that the enzyme is first immobilizedon the electrode that has been prefabricated without enzyme. As aconsequence, there is the problem that the enzyme cannot be coupled tothe electrode components in a controlled manner. Thus, whenglutaraldehyde is used as a cross-linking reagent, the enzyme not onlybinds in an uncontrolled manner to any reactive components of theelectrode material, but it is also inter-crosslinked. Furthermore, thisprocedure contaminates the electrode with the reagents that are usedand, hence, the electrode has to again be thoroughly cleaned especiallybefore use in an in vivo biosensor which increases the productioncomplexity and thus the costs.

Cho et al. (Biotechnology and Bioengineering (1977), 19, 769-775)describe the immobilization of enzymes on activated carbons by covalentcoupling. More particularly, the immobilization of glucose oxidase topetroleum-based activated carbons by (a) adsorption of the enzymefollowed by cross-linking with glutaraldehyde or (b) activation of thecarbon surface with a diimide and subsequent reaction with the enzyme isdescribed. By this means, a considerably slower deactivation of theenzyme in the presence of H₂O₂ could be shown as compared to the solubleenzyme.

Li et al. (Analytical and Bioanalytical Chemistry (2005), 383, 918-922)disclose a glucose biosensor comprising a modified glassy carbonelectrode as the working electrode. The modified glassy carbon electrodeis prepared by coating a commercial electrode's surface with adispersion of functionalized multi-wall carbon nanotubes (MWCNTs) havingoxidized glucose oxidase covalently attached thereto in PBS buffersolution which contains Nafion® and ferrocene monocarboxylic acid. Thecatalytic effect of the functionalized nanotubes for glucose oxidationis particularly emphasized.

US 2007/0029195 A1 discloses immobilization of biomolecules such asproteins by covalent coupling to a conductive polymeric matrixreinforced by nanoparticles to improve the mechanical stability,electrical conductivity and immobilization of biomolecules. The matrixis a nanocomposite comprising gold nanoparticles coated with apolypyrrole formed from pyrrole and pyrrole propylic acid, wherein thelatter compound provides for the covalent attachment of thebiomolecules.

US 2008/0014471 A1 discloses electrochemical sensors comprisingelectrodes employing cross-linked enzyme clusters immobilized on carbonnanotubes (CNTs). In detail, the immobilization involvesfunctionalization of the nanotubes' surface, subsequent covalentattachment of an enzyme such as glucose oxidase by means of a linkingreagent to yield a CNT-enzyme conjugate, precipitation of free enzymeswith a precipitation agent, and final treatment with a cross-linkingreagent to form cross-linked enzyme clusters covalently attached to theCNTs via the CNT-enzyme conjugate.

US 2008/044911 A1 discloses a glucose sensor comprising nanowires havingglucose oxidase covalently attached to their surface, whichfunctionalized nanowires are prepared by contacting conventionalnanowires with a linking reagent, e.g. a silane, and the enzyme.Conversion of glucose in a sample to be examined by the glucose oxidaseimmobilized on the nanowires' surface results in a change in pH of thetest solution, wherein this change in pH produces a signal in thenanowires which can be detected by suitable means.

It is known in the art that covalent coupling of an enzyme to a support(e.g. MANAE-agarose, activated glyoxyl agarose and glutaraldehydeagarose) leads to a stabilization of the enzyme against thermaldecomposition (Betancor et al., Journal of Biotechnology (2006), 121,284-289). However, in addition to thermal decomposition, biosensorsemploying such immobilized enzymes also face the problem of enzymedecomposition caused by organic solvents during storage of the biosensoror caused by oxidative agents such as H₂O₂ during the period ofoperation.

In practice, biosensors must fulfill a plurality of requirements inorder to allow exact measurements for immediate or time-delayedtherapeutic measures. In particular, it is of uppermost importance thatthe analyte of interest can be determined with both a high specificityand sensitivity in order to enable determination of low amounts of theclinically relevant parameter. Consequently, the significant loss inenzymatic activity generally observed in commercially availablebiosensors when storing the same for more than one month is notacceptable for diagnostical and/or clinical purposes.

SUMMARY

Hence, an object of the invention is to provide a composition forforming an electrode, in which the disadvantages of the prior art are atleast partially eliminated. In particular, the composition should ensurea specific and durable immobilization of the enzyme, prevent or at leastreduce the loss of enzymatic activity upon production of the electrodecomposites, storage for a period of more than one month as well asbiosensor function, and guarantee a high sensitivity over the wholeperiod of operation.

The invention provides compositions for forming an electrode, thecompositions comprising an electrically conductive component having ananalyte-specific enzyme covalently attached thereto, wherein thecomposition further comprises at least one electrically nonconductive orsemiconductive enzyme-stabilizing component.

According to the invention, the composition comprises an electricallyconductive component having an analyte-specific enzyme covalentlyattached thereto. The term “electrically conductive component” as usedwithin the present application refers to any material containingfree-moveable electric charges and having a resistivity of ρ<10⁻⁴ Ωcm,thereby allowing the transport of electric current. The electricallyconductive material may be an electronic conductor (first orderconductor) or an ion conductor (second order conductor), with anelectronic conductor being preferred.

Preferably, the electrically conductive component is aH₂O₂-decomposition catalyst. This means that the electrically conductivecomponent does not only transport electric current, but is additionallyable to catalyze the decomposition of hydrogen peroxide which is presentor formed in a sample contacting the electrically conductive component.Since hydrogen peroxide generally causes damage to most of the enzymesused in the detection of clinically relevant analytes and additionallycan act as an inhibitor of the analyte or of co-substrates such asoxygen, the decomposition of hydrogen peroxide in biochemical testelements is of crucial importance. For this purpose, the electricallyconductive component may, for example, catalyze the chemical conversionof hydrogen peroxide to a chemically less active or inert substance,including the oxidation of hydrogen peroxide to water.

In another preferred embodiment of the present invention, theelectrically conductive component is selected from the group consistingof activated carbon, carbon black, graphite, carbonaceous nanotubes,palladium, platinum, and hydroxides of iron oxide such as for exampleFeO(OH), with graphite and carbonaceous nanotubes being especiallypreferred.

The term “carbonaceous nanotubes” as used herein refers to all kinds ofnanotubes; i.e., tubes having an average inner diameter of <1 μm,comprising carbon as one of their components. In particular,carbonaceous nanotubes in the sense of the present invention comprisecarbon nanotubes (CNT) which may be in the form of, for example,single-wall carbon nanotubes (SWCNT), double-wall carbon nanotubes(DWCNT), multi-wall carbon nanotubes (MWCNT), etc. Since nanotubesgenerally provide a high effective surface, they can be extensivelymodified with suitable substances to be immobilized on their surface,such as for example enzymes employed in the detection of an analyte.

In another embodiment, the composition according to the invention maycomprise at least one additional electrically conductive component inaddition to the electrically conductive component having theanalyte-specific enzyme covalently attached thereto. The additionalelectrically conductive component may be any material capable oftransporting electric current and is preferably selected from the groupconsisting of activated carbon, carbon black, graphite, carbonaceousnanotubes, palladium, platinum, and hydroxides of iron oxide. Thiscomponent may, in principle, also form a conjugate with theanalyte-specific enzyme by means of covalent bonds, but preferably isnot covalently attached to the analyte-specific enzyme.

According to the invention, the electrically conductive component has ananalyte-specific enzyme covalently attached thereto. The enzyme may beany enzyme which is specific for the analyte to be detected and whichappears to be suitable to a person skilled in the art for achieving thedesired effect. The enzyme immobilized on the electrically conductivecomponent is preferably an oxidase and in particular alcohol oxidase(1.1.3.13), arylalcohol oxidase (EC 1.1.3.7), catechol oxidase (EC1.1.3.14), cholesterol oxidase (EC 1.1.3.6), choline oxidase (1.1.3.17),galactose oxidase (EC 1.1.3.9), glucose oxidase (EC 1.1.3.4),glycerol-3-phosphate oxidase (EC 1.1.3.21), hexose oxidase (EC 1.1.3.5),malate oxidase (EC 1.1.3.3), pyranose oxidase (EC 1.1.3.10),pyridoxine-4-oxidase (EC 1.1.3.12) or thiamine oxidase (EC 1.1.3.23).More preferably, the enzyme is glucose oxidase.

Preferably, the analyte-specific enzyme is selectively covalently boundto the electrically conductive component. The covalent binding of theenzyme to the electrically conductive component ensures the constancy ofthe function of an electrode comprising the inventive compositionbecause a detachment of enzyme can be ruled out under the typicalmeasurement conditions (physiological electrolyte concentration,physiological pH, body temperature). Thus, electrochemical sensorscomprising such electrodes remain operational over a long time periodand virtually operate free of drift.

In order to covalently bind the analyte-specific enzyme to theelectrically conductive component, the present invention envisages in apreferred embodiment that the electrically conductive component has asurface comprising functional groups to which the enzyme is bound. Thesurface can, for example, exhibit hydroxy, carboxy or aminofunctionalities. Alternatively, the surface can be functionalized bycoating the electrically conductive component with a suitable reagent toform functional groups by means of which the enzyme can be covalentlybound to the surface of the electrically conductive component.

Coating reagents which are used within the scope of the presentinvention are substances which, on the one hand, undergo a covalentbinding with the electrically conductive component and, on the otherhand, contain at least one functional group which serves to covalentlybind the enzyme. This means that the coating reagents are at leastbifunctional; i.e., comprise at least two functional groups which may bethe same or different.

In a preferred embodiment, the enzyme is directly covalently bound tothe surface of the electrically conductive component, which means thatat least one covalent bond is formed between a functional group of theenzyme and a functional group on the surface of the electricallyconductive component. The enzyme can be coupled to the surface in anymanner and can comprise a prior activation of functional groups on thesurface of the electrically conductive component and/or of the enzyme.

Functional groups can for example be activated by reacting thefunctionalized electrically conductive component and/or the enzyme witha suitable activation reagent. Preferred activation reagents comprisecarbodiimides such as for example dicyclohexylcarbodiimide (DCC),diisopropylcarbodiimide or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide(EDC) as well as combinations of carbodiimides and succinimides. Aparticularly suitable activation reagent for the purposes of the presentinvention comprises a combination of1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) andN-hydroxysuccinimide (NHS).

Other methods suitable for covalently binding, in particular selectivelycovalently binding the analyte-specific enzyme to the electricallyconductive component are known to a person skilled in the art and aredescribed, for example, in H. Weetall, Methods of Enzymology (1976), 33,134-148.

According to the invention, the composition further comprises anelectrically nonconductive or semiconductive enzyme-stabilizingcomponent. The term “electrically nonconductive component” as usedwithin the present application refers to any material having aresistivity of ρ>10⁹ Ω cm and incapable of transporting electriccurrent. In contrast, the term “electrically semiconductive component”as used herein refers to any material having a resistivity of 10⁻⁴?ρ?10⁹Ωcm. The term “enzyme-stabilizing” means that the nonconductive orsemiconductive component is able to stabilize the analyte-specificenzyme by either reacting with the analyte-specific enzyme to form acomplex or conjugate with the enzyme or by converting chemicalsubstances causing decomposition of the enzyme.

In a preferred embodiment, the electrically nonconductive orsemiconductive enzyme-stabilizing component is a H₂O₂-decompositioncatalyst which means that the electrically nonconductive orsemiconductive enzyme-stabilizing component decomposes hydrogenperoxide, for example by chemical conversion, in order to prevent adamage to the analyte-specific enzyme employed in the compositionaccording to the present invention.

The electrically nonconductive or semiconductive enzyme-stabilizingcomponent is preferably selected from the group consisting of anH₂O₂-degrading metal oxide and an H₂O₂-degrading enzyme. For the purposeof the present invention the H₂O₂-degrading metal oxide may be any metaloxide which is able to catalyze the decomposition of hydrogen peroxidefor example by oxidizing the same, with Ag₂O, Al₂O₃ or an oxide of ametal of the 4th period of the periodic table having proven to beparticularly advantageous. The oxide of a metal of the 4th period of theperiodic table is preferably an oxide of Mn, CuO or ZnO, wherein MnO₂,Mn₃O₄ or Mn₅O₈ are especially preferred. The H₂O₂-degrading enzyme maybe any enzyme known to decompose hydrogen peroxide and particularencompasses a peroxidase and catalase.

In another preferred embodiment, the composition according to theinvention further comprises the analyte-specific enzyme covalentlyattached to the electrically nonconductive or semiconductiveenzyme-stabilizing component. This means that the analyte-specificenzyme employed in the inventive composition may not only be covalentlyattached to the electrically conductive component but can additionallybe covalently attached to the electrically nonconductive orsemiconductive enzyme-stabilizing component by means of covalent bondsformed between the enzyme and, for example, functional groups on thesurface of the electrically nonconductive or semiconductiveenzyme-stabilizing component.

The electrically conductive component and/or the electricallynonconductive or semiconductive enzyme-stabilizing component may beprovided according to the invention in a particulate form, wherein theparticle size can be varied depending on the respective requirements. Ina preferred embodiment, the composition according to the inventioncomprises the electrically conductive component and/or the electricallynonconductive or semiconductive enzyme-stabilizing component in the formof nanoparticles; i.e., particles having an average diameter of <1 μm.Within the scope of the present invention 90% of the nanoparticlesusually have a diameter of 10 nm to 100 nm, a diameter of 15 nm to 30 nmhaving proven to be particularly preferred.

The ability to control the effective surface of the electricallyconductive component and/or the electrically nonconductive orsemiconductive enzyme-stabilizing component by means of the particlesize is of crucial importance, for example, with respect to afunctionalization with chemical substances. More particularly, a highereffective surface can increase the loading with enzyme and, thus, canresult in a higher enzyme activity stated in units per milligram ofenzyme-loaded component. The electrically conductive component used forthe purposes of the present invention usually has an enzyme activity ofabout 10 mU/mg to about 5 U/mg, with an enzyme activity of about 100mU/mg to about 1 U/mg having proven to be particularly advantageous. Theterm “unit” as used within the scope of the present applicationrepresents the amount of enzyme which is required to convert 1 μmolsubstrate per minute under standard conditions.

In addition to the electrically conductive component and theelectrically nonconductive or semiconductive enzyme-stabilizingcomponent the composition according to the invention may comprise othercomponents conventionally employed for forming an electrode such as, forexample, binders, fillers and the like. As regards binders, thecomposition preferably comprises at least one binder selected from thegroup consisting of fluorinated hydrocarbons such as Teflon®,polycarbonates, polyisoprene, polyurethanes, acrylic resins, polyvinylresins and silicones, with polyurethanes, acrylic resins and polyvinylresins being more preferred.

For forming an electrode, the electrically conductive component havingthe analyte-specific enzyme covalently attached thereto, theelectrically nonconductive or semiconductive enzyme-stabilizingcomponent and all other components required to form an electrode matrixare thoroughly mixed and subsequently dried such that the electricallyconductive component and/or the electrically nonconductive orsemiconductive enzyme-stabilizing component are preferably homogeneouslydispersed in the composition.

Depending on the specific requirements of the electrode to be formed,the skilled person is able to determine the amounts of the differentcomponents required for providing a composition according to theinvention without any difficulty. As regards the electrically conductivecomponent, however, it has been proven as advantageous when thecomposition contains the conjugate formed from the electricallyconductive component and the analyte-specific enzyme in an amount offrom 0.5 to 10% by weight, based on the dry weight of the composition.On the other hand, the composition preferably contains the electricallynonconductive or semiconductive enzyme-stabilizing component in anamount of from 5 to 50% by weight, based on the dry weight of thecomposition.

The composition described herein significantly reduces the loss ofactivity of analyte-specific enzyme required for detecting an analyte ofinterest by means of a biosensor such as, for example, anelectrochemical sensor encompassing an electrode making use of thecomposition according to the invention. In particular, in thecomposition according to the invention the enzyme has a residualactivity of at least 90% after storing the composition for at least 4weeks, preferably at least 12 weeks, more preferably at least 28 weeksat a temperature of 4° C., based on the total activity of the enzymebefore storage; i.e., based on the total activity of the enzyme in thecomposition directly after its preparation.

In another aspect, the present invention thus relates to anelectrochemical sensor for determining an analyte, the sensor comprisingat least one working electrode and at least one reference electrode,wherein the working electrode comprises the composition according to theinvention.

Whereas the working electrode of the electrochemical sensor according tothe invention serves to convert the analyte to be determined, thereference electrode allows to adjust the polarization potential of theworking electrode and can consist of any material which is suitable forthe purposes of the invention. A metal/metal ion electrode, inparticular a silver/silver chloride electrode, is preferably used as thereference electrode. In addition to the at least one working electrodeand the at least one reference electrode, the electrochemical sensor mayalso comprise at least one counter electrode which is preferably in theform of a noble metal electrode, in particular a gold electrode, or agraphite electrode.

According to the invention, the electrochemical sensor preferablycomprises a biocompatible coating covering the at least one workingelectrode, the at least one reference electrode and optionally thecounter electrode(s). The biocompatible coating allows the analyte topenetrate into the electrode matrix but should prevent electrodecomponents from escaping into the surrounding medium containing theanalyte of interest.

In view of the fact that due to the covalent binding of the enzyme tothe electrically conductive component the enzyme does not bleed out ofthe working electrode or the electrochemical sensor, a biocompatiblecoating is not absolutely necessary for many applications. Thus, theelectrochemical sensor according to the invention can also be used in invivo applications when the biocompatible coating is not a barrier toenzymes. In such case, a biocompatible coating can be selected whichprovides an optimal interaction with the surrounding tissue and/or bloodor serum.

Biocompatible coatings can be generated in various ways. A preferredmethod is to use prefabricated membranes which are applied to theelectrochemical sensor. The membrane can be immobilized on the sensor byvarious techniques, with gluing or laser welding being regarded aspreferred. Alternatively, the biocompatible coating can be generated insitu by applying a solution of a suitable polymer onto theelectrochemical sensor and subsequently drying it. The application ofthe polymer onto the biosensor is preferably carried out by spraying,dip-coating or dispersing a dilute solution of the polymer in alow-boiling organic solvent, but is not limited to these methods.

Polymers which are suitable for such purposes comprise in particularpolymers having a zwitterionic structure and mimicking cell surfacessuch as for example2-methacryloyloxyethyl-phosphorylcholine-co-n-butyl-methacrylate(MPC-co-BMA). The biocompatible coatings that are obtained usually havea thickness of about 1 μm to about 100 μm, preferably of about 3 μm toabout 25 μm.

The electrochemical sensor according to the invention is preferablydesigned for multiple measurements; i.e., the sensor enables a repeatedmeasurement of the analyte to be determined. This is particularlydesirable in applications in which a constant, i.e., continuous ordiscontinuous control of the presence and/or the amount of an analyte isto take place over a longer time period of, e.g., one day or longer, inparticular one week or longer. In a particularly preferred embodiment,the electrochemical sensor according to the invention thus is fully orpartially implantable and can be implanted, for example, into fattytissue or into blood vessels. Alternatively, the invention allows theelectrochemical sensor to be designed as a flow-through cell throughwhich a fluid sample containing the analyte is passed.

The electrochemical sensor described herein can be used to determine ananalyte in a fluid medium which can originate from any source. In apreferred embodiment, the electrochemical sensor is used to determine ananalyte in a body fluid comprising but not limited to whole blood,plasma, serum, lymph fluid, bile fluid, cerebrospinal fluid,extracellular tissue fluid, urine as well as glandular secretions suchas saliva or sweat, wherein whole blood, plasma, serum and extracellulartissue fluid are regarded as particularly preferred. The amount ofsample required to carry out the analysis is usually from about 0.01 μlto about 100 μl, preferably from about 0.1 μl to about 2 μl.

The analyte to be determined qualitatively and/or quantitatively can beany biological or chemical substance which can be detected by means of aredox reaction. The analyte is preferably selected from the groupconsisting of malic acid, alcohol, ammonium, ascorbic acid, cholesterol,cysteine, glucose, glutathione, glycerol, urea, 3-hydroxybutyrate,lactic acid, 5′-nucleotidase, peptides, pyruvate, salicylate andtriglycerides. In a particularly preferred embodiment, the analyte to bedetermined by means of the electrochemical sensor according to theinvention is glucose.

In yet a further aspect, the present invention relates to a method fordetermining an analyte, comprising the steps:

(a) contacting a sample containing the analyte with an electrochemicalsensor according to the invention, and

(b) determining the presence and/or the amount of the analyte.

In order to determine the analyte, the electrochemical sensor can bedesigned in any manner which allows a contact between theelectrochemical sensor and the sample containing the analyte. Thus, thesensor can be designed as a flow-through cell through which the mediumcontaining the analyte flows. On the other hand, the sensor can also bedesigned as a diffusion sensor, wherein the contact between the sensorand medium takes place by diffusion. Equally, the electrochemical sensorcan be designed as a device which is intended to be completely orpartially implanted into the body of a patient, in which case it isimplanted either into a blood vessel or into tissue and in particularinto subcutaneous fatty tissue.

A measurable signal is generated by the sensor depending on the presenceand/or the amount of analyte. This signal is preferably an electricalsignal such as for example electrical current, voltage, resistance etc.which is evaluated or read-out using suitable means. The electrochemicalsensor is preferably an amperometric sensor.

DRAWINGS

It is intended to further elucidate the invention by the followingfigures and examples.

FIG. 1 shows a measuring signal [nA] of an electrochemical sensoraccording to the present invention plotted against time [s] independence on the glucose concentration [0, 2, 4, 6, 8, 12, 17, 23, 26,20, 15, 10, 7, 5, 3, 0.8 mM], the sensor employing a working electrodecomprising a conjugate formed from carbon nanotubes and glucose oxidase(CNT-GOD; 1.75% by weight) and MnO₂ (20% by weight) as semiconductiveH₂O₂-decomposition catalyst. Polarisation voltage 350 mV.

FIG. 2 shows a measuring signal [nA/mM] of an electrochemical sensoraccording to the present invention stored over a period of 28 weeks at atemperature of 4° C., the sensor employing a working electrodecomprising a conjugate formed from carbon nanotubes and glucose oxidase(CNT-GOD; 2.4% by weight) and MnO₂ (19.5% by weight) as semiconductiveH₂O₂-decomposition catalyst.

FIG. 3 shows enzymatic activity [mU/mg lyophilized conjugate] of aconjugate formed from carbon nanotubes and glucose oxidase (CNT-GOD) inMES buffer (pH 7.4), in MES buffer in the presence of H₂O₂ (5%solution), and in MES buffer in the presence of H₂O₂ (5% solution) and500 U/ml of catalase.

FIGS. 4A, 4B, and 4C show a decomposition rate of H₂O₂ [mM/mg/h] causedby different electrically conductive and electrically nonconductive orsemiconductive materials:

FIG. 4A shows a decomposition rate of H₂O₂ [mM/mg/h] caused by differentcommercially available carbons.

FIG. 4B shows a decomposition rate of H₂O₂ [mM/mg/h] caused by differentcommercially available metal oxides.

FIG. 4C shows a decomposition rate of H₂O₂ [mM/mg/h] caused by twodifferent manganese oxides.

FIGS. 5A and 5B show a measuring signal [nA] of differentelectrochemical sensors plotted against increasing glucoseconcentrations [mg/dl]. Polarisation voltage 350 mV:

FIG. 5A shows a measuring signal [nA] of 8 electrochemical sensorsemploying a working electrode comprising a conjugate formed from carbonnanotubes and glucose oxidase (CNT-GOD; 2% by weight).

FIG. 5B shows a measuring signal [nA] of 8 electrochemical sensorsaccording to the present invention, the sensors employing a workingelectrode comprising a conjugate formed from carbon nanotubes andglucose oxidase (CNT-GOD; 2% by weight) and MnO₂ (20% by weight) assemiconductive H₂O₂-decomposition catalyst.

FIG. 6 shows a schematic representation of an electrochemical sensoraccording to the present invention.

DETAILED DESCRIPTION

The following description of technology is merely exemplary in nature ofthe subject matter, manufacture and use of one or more inventions, andis not intended to limit the scope, application, or uses of any specificinvention claimed in this application or in such other applications asmay be filed claiming priority to this application, or patents issuingtherefrom.

EXAMPLES Example 1 Preparation of a Conjugate Formed From CarbonNanotubes and Glucose Oxidase (CNT-GOD)

For preparing an electrically conductive component having an enzymecovalently attached thereto, 2.5 g of carbon nanotubes (CNT, Nanolab)were added to a solution of 480 mg glucose oxidase (GOD, Roche), 9.6 gof 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC, Sigma), and 7.2g of N-hydroxysuccinimide (NHS, Sigma) in 480 ml of Millipore water.After incubating the mixture for 6 hours at room temperature on alaboratory shaker, the mixture was passed through a membrane filter,thereby separating the conjugate from the reaction solution. Theconjugate was washed with PBS buffer (3×120 ml), Millipore water (1×120ml), and dried overnight under vacuum, yielding 3.0 g of CNT-GODconjugate.

Example 2 Determination of the Enzymatic Activity of the CNT-GODConjugate

In order to determine the enzymatic activity of the CNT-GOD conjugateprepared in Example 1, 5 mg of lyophilized conjugate were suspended in 1ml of MES buffer and stirred for 1 hour at room temperature. Inparallel, Toyobo solution (GLO-201; 30 ml of MES buffer (79 mM); 6 ml ofglucose solution (131 mM); 0.3 ml of 4-aminoantipyrine solution (0.2mM); 0.3 ml of N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine solution(0.3 mM); 0.3 ml of peroxidase solution (about 4 U/ml)) was pipettedinto a cuvette and tempered for 10 minutes at 37° C. After adding theconjugate to the tempered Toyobo solution, absorption of the mixturethus obtained was measured using a wavelength of 555 nm. By this means,the enzymatic activity was determined to be 870 mU/mg conjugate.

Example 3 Stability of an Electrochemical Sensor Employing A WorkingElectrode Comprising A CNT-GOD Conjugate and a H₂O₂-DecompositionCatalyst

In order to determine the stability of an electrochemical sensoraccording to the present invention, a working electrode comprising anelectrode matrix consisting of 78.25% by weight of an electrode paste(Acheson), 1.75% by weight of the CNT-GOD conjugate of Example 1 and 20%by weight of MnO₂ (Merck) was prepared.

Subsequently, the electrochemical sensor was contacted with a solutioncontaining glucose at concentrations of 0, 2, 4, 6, 8, 12, 17, 23, 26,20, 15, 10, 7, 5, 3 and 0.8 mM periodically varying over a period of 5days. As can be derived from FIG. 1, the electrochemical sensor did notshow any loss of sensitivity over the whole period which is a result ofboth prevention of enzyme bleeding caused by covalent binding to thecarbon nanotubes and the decomposition of H₂O₂ catalyzed by MnO₂.

Example 4 Long-Term Stability of an Electrochemical Sensor Employing aWorking Electrode Comprising a CNT-GOD Conjugate and aH₂O₂-Decomposition Catalyst

In order to determine the long-term stability of an electrochemicalsensor according to the present invention, a working electrodecomprising an electrode matrix consisting of 78% by weight of anelectrode paste (Acheson), 2.4% by weight of the CNT-GOD conjugate ofExample 1 and 19.5% by weight of MnO₂ (Merck) was prepared.

Subsequently, the electrochemical sensor was stored over a period of 28weeks at a temperature of 4° C. As becomes evident from FIG. 2, themeasuring signal of the electrochemical sensor remains unchanged betweenweek 4 and week 28 following its preparation. The loss of sensitivitybetween week 0 and week 4 is ascribed to conditioning effects of theelectrode matrix.

Example 5 Determination of the Enzymatic Activity of a CompositionComprising a CNT-GOD Conjugate and a H₂O₂-Decomposition Catalyst

In order to determine the enzymatic activity of a composition accordingto the present invention, 10 mg of a lyophilized CNT-GOD-conjugate weresuspended in 1 ml of MES buffer (pH 7.4) and incubated for 1 hour atroom temperature.

Subsequently, a commercially available H₂O₂ assay (Toyobo) was used tomeasure the residual enzymatic activity of the CNT-GOD conjugate, whichwas determined to be about 600 mU/mg lyophilized conjugate (cf. FIG. 2).In parallel, both the residual enzymatic activity of the CNT-GODconjugate in MES buffer in the presence of H₂O₂ (5% solution) and theresidual enzymatic activity of CNT-GOD in MES buffer in the presence ofH₂O₂ (5% solution) and 500 U/ml of catalase were determined.

As can be seen from FIG. 3, the residual enzymatic activity of theCNT-GOD conjugate decreased to less than 100 mU/mg lyophilized conjugatewhen H₂O₂ was present in the sample (CNT-GOD+H₂O₂). On the other hand,the addition of both H₂O₂ solution and the H₂O₂-decomposition catalystcatalase resulted in a residual enzymatic activity of about 650 mU/mglyophilized conjugate (CNT-GOD+H₂O₂+catalase), indicating completerepression of denaturation of GOD by H₂O₂.

Example 6 Determination of H₂O₂-Decomposition Catalysts

In order to determine compounds acting as H₂O₂-decomposition catalysts,a number of carbons and metal oxides have been evaluated with respect totheir ability to effectively catalyze the decomposition of H₂O₂.

In detail, Carbon Black Acetylene (Strem Chemicals), Spheron 6400 (CabotCorporation), Mogul E (Cabot Corporation), Carbon Black Acetylene (AlfaAesar), Mogul L (Cabot Corporation), Nanofibers (Electrovac), VulcanBlack XC-605 (Cabot Corporation), Black Pearls 2000 (Cabot Corporation;ground for 60 min), Black Pearls 2000 (Cabot Corporation; ground for 120min) and Nanotubes (Nanolab) were tested as electrically conductivecarbon materials. In this context, carbon nanotubes (Nanolab) and BlackPearls 2000 (Cabot Corporation; ground for 120 min) were shown to beparticularly suitable, providing a H₂O₂-decomposition rate of 0.00126and 0.00098 mM/mg catalyst/h, respectively (cf. FIG. 4A).

As regards the metal oxides, it becomes clear from FIGS. 4B and 4C thatthe different compounds employed in the test provide quite differentdecomposition rates. Whereas the catalytic effect of commerciallyavailable Al₂O₃ (Sigma-Aldrich, Product No. 229423), Al₂O₃ (Aldrich,Product No. 551643) and FeO(OH) (Fluka, Product No. 71063) was ratherpoor, Ag₂O (Riedel-de Haen, Product No. 10228), and Mn₃O₄ (StremChemicals, Product No. 93-2513) were shown to be very effective asH₂O₂-decomposition catalysts (cf. FIG. 4B).

Another compound very effectively catalyzing the decomposition of H₂O₂is Mn₅O₈ which was obtained by heating commercially available Mn₃O₄(Strem Chemicals, Product No. 93-2513) in 6 hours from 25° C. to 430°C., holding this temperature for 12 hours, and subsequently cooling to25° C. As can be derived from FIG. 4C, the decomposition rate of H₂O₂observed in the presence of Mn₅O₈ obtained by the above procedure wassignificantly better than the catalyzing effect of Mn₃O₄.

Example 7 Reduction of Overpotential in an Electrochemical SensorEmploying a Working Electrode Comprising a CNT-GOD Conjugate and aH₂O₂-Decomposition Catalyst

In order to determine the effect of an electrically nonconductive orsemiconductive enzyme-stabilizing component on the overvoltage observedat the working electrode of electrochemical sensors, eightelectrochemical sensors employing a working electrode comprising 2% byweight of the CNT-GOD conjugate of Example 1 and eight electrochemicalsensors employing a working electrode comprising both 2% by weight ofthe CNT-GOD conjugate of Example 1 and 20% by weight of MnO₂ (Merck)were prepared.

As becomes evident from FIG. 5A, the use of working electrodescomprising the CNT-GOD conjugate alone resulted in a low sensitivity of0.01±0.03 nA/mM glucose solution when applying a polarisation voltage of350 mV (relative to an Ag/AgCl reference electrode) and measuring theelectric current in dependence on the glucose concentration.

In contrast, the use of working electrodes comprising the CNT-GODconjugate in combination with MnO₂ resulted in a remarkably increasedsensitivity of 0.54±0.11 nA/mM glucose solution under the same reactionconditions, indicating a significant reduction of overvoltage at theworking electrode (cf. FIG. 5B).

What is claimed is:
 1. A composition for forming an electrode, thecomposition comprising: an electrically conductive component; anelectrically nonconductive or semiconductive component; and ananalyte-specific enzyme, wherein the analyte specific enzyme iscovalently bonded only to the electrically conductive component, andwherein the electrically nonconductive or semiconductive componentstabilizes the analyte specific enzyme.
 2. The composition according toclaim 1, wherein the electrically conductive component is aH₂O₂-decomposition catalyst.
 3. The composition according to claim 1,wherein the electrically conductive component is selected from the groupconsisting of activated carbon, carbon black, graphite, carbonaceousnanotubes, palladium, platinum, and hydroxides of iron oxide.
 4. Thecomposition according to claim 3, wherein the electrically conductivecomponent is selected from the group consisting of graphite andcarbonaceous nanotubes.
 5. The composition according to claim 1, whereinthe enzyme is an oxidase.
 6. The composition according to claim 5,wherein the enzyme is a glucose oxidase.
 7. The composition according toclaim 1, wherein the at least one electrically nonconductive orsemiconductive component comprises a H₂O₂-decomposition catalyst.
 8. Thecomposition according to claim 1, wherein the at least one electricallynonconductive or semiconductive component is selected from the groupconsisting of an H₂O₂-degrading metal oxide and an H₂O₂-degradingenzyme.
 9. The composition according to claim 8, wherein the at leastone electrically nonconductive or semiconductive component comprisesAg₂O, Al₂O₃ or an oxide of a metal of the 4th period of the periodictable.
 10. The composition according to claim 8, wherein the at leastone electrically nonconductive or semiconductive component comprisesperoxidase or catalase.
 11. The composition according to claim 9,wherein the oxide of a metal of the 4th period of the periodic table isMnO₂, Mn₃O₄ or Mn₅O₈, CuO or ZnO.
 12. The composition according to claim1, wherein the electrically conductive component and/or the electricallynonconductive or semiconductive enzyme-stabilizing component is providedin the form of nanoparticles.
 13. The composition according to claim 1,further comprising at least one binder selected from the groupconsisting of fluorinated hydrocarbons, polycarbonates, polyisoprene,polyurethanes, acrylic resins, polyvinyl resins and silicones.
 14. Thecomposition according to claim 1, wherein the electrically conductivecomponent and/or the electrically nonconductive or semiconductiveenzyme-stabilizing component is homogeneously dispersed in thecomposition.
 15. The composition according to claim 1, wherein theenzyme has a residual activity of at least 90% after storing thecomposition for at least 4 weeks at a temperature of 4° C., based on thetotal activity of the enzyme before storage.
 16. The compositionaccording to claim 15, wherein the enzyme has a residual activity of atleast 90% after storing the composition for at least 28 weeks at atemperature of 4° C., based on the total activity of the enzyme beforestorage.
 17. An electrochemical sensor for determining an analyte, thesensor comprising at least one working electrode and at least onereference electrode, wherein the working electrode comprises thecomposition according to claim
 1. 18. The electrochemical sensoraccording to claim 17, wherein it comprises a biocompatible coatingcovering the working electrode and the reference electrode.
 19. Theelectrochemical sensor according to claim 17 for determining an analytein a body fluid.
 20. The electrochemical sensor according to claim 17for determining an analyte selected from the group consisting of malicacid, alcohol, ammonium, ascorbic acid, cholesterol, cysteine, glucose,glutathione, glycerol, urea, 3-hydroxybutyrate, lactic acid,5′-nucleotidase, peptides, pyruvate, salicylate and triglycerides. 21.The electrochemical sensor according to claim 20 for determiningglucose.
 22. A method for determining an analyte, comprising the steps:(a) contacting a sample containing the analyte with an electrochemicalsensor according to claim 17, and (b) determining the presence and/orthe amount of the analyte.
 23. A composition for forming an electrode,the composition comprising: an electrically conductive componentcovalently bonded to an analyte-specific enzyme, and an electricallynonconductive or semiconductive component, wherein only the electricallyconductive component is covalently bonded to the analyte-specificenzyme.